Team:Lethbridge/Notebook/Lab Work/April

From 2010.igem.org

(Difference between revisions)
(New page: April)
 
(21 intermediate revisions not shown)
Line 1: Line 1:
-
April
+
<div style="background-color:#000000; color:white">
 +
<html>
 +
 
 +
<br>
 +
 
 +
<table border="0" width="100%" style="background-color:#000000">
 +
 
 +
<tr>
 +
 
 +
<th>
 +
 
 +
<image src="https://static.igem.org/mediawiki/2010/2/29/UofLteamlogo.jpg" width="200px"/>
 +
 
 +
</th>
 +
 
 +
<th>
 +
 
 +
<image src="https://static.igem.org/mediawiki/2010/9/91/UofLLabWork.JPG" height="300px"/>
 +
 
 +
</th>
 +
 
 +
<th>
 +
 
 +
<image src="https://static.igem.org/mediawiki/2010/2/29/UofLteamlogo.jpg" width="200px"/>
 +
 
 +
</th>
 +
 
 +
</tr>
 +
 
 +
</table>
 +
 
 +
 
 +
 
 +
<br>
 +
 
 +
<align="centre">
 +
<table border="0"  width="100%"  style="background-color:#000000">
 +
 
 +
<tr>
 +
 
 +
<th>
 +
 
 +
<a href="https://2010.igem.org/Team:Lethbridge">
 +
<img src="https://static.igem.org/mediawiki/2010/2/22/UofLHome.jpg" width="80"/>
 +
</a>
 +
 
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Team">
 +
<img src="https://static.igem.org/mediawiki/2010/0/0d/UofLTeam.jpg" width="80"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Project">
 +
<img src="https://static.igem.org/mediawiki/2010/8/8d/UofLProjectbutton.jpg" width="80"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work">
 +
<img src="https://static.igem.org/mediawiki/2010/7/73/UofLNotebookbutton.jpg" width="80"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Parts">
 +
<img src="https://static.igem.org/mediawiki/2010/8/84/UofLPartsSubmittedToTheRegistrybutton.jpg" width="80"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Modeling">
 +
<img src="https://static.igem.org/mediawiki/2010/e/e1/UofLModelingbutton.jpg" width="80"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics">
 +
<img src="https://static.igem.org/mediawiki/2010/2/26/UofLEthicsbutton.jpg" width="80"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Safety">
 +
<img src="https://static.igem.org/mediawiki/2010/0/00/UofLSafetybutton.jpg" width="80"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Art">
 +
<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/News">
 +
<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLNewsButton.jpg" width="80"/>
 +
</a>
 +
</th>
 +
</table>
 +
</body>
 +
</html>
 +
<hr>
 +
<html>
 +
<center>
 +
<font color="white">Feel free to look around our notebook!
 +
</center>
 +
</html>
 +
 
 +
<html>
 +
<body>
 +
<center>
 +
<table border="0"  width="28%"  style="background-color:#000000">
 +
 
 +
<tr>
 +
<th>
 +
<div class="miniBar">
 +
<div class="countdown"><object type="application/x-shockwave-flash" data="http://www.oneplusyou.com/bb/files/countdown/countdown.swf?co=FFFFFF&bgcolor=000000&date_month=10&date_day=27&date_year=0&un=THE WIKI FREEZE&size=normal&mo=10&da=27&yr=2010" width="300" height="100"><param name="movie" value="http://www.oneplusyou.com/bb/files/countdown/countdown.swf?co=FFFFFF&bgcolor=000000&date_month=10&date_day=27&date_year=0&un=THE WIKI FREEZE&size=normal&mo=10&da=27&yr=2010" /><param name="bgcolor" value="#000000" /></object><img src="http://www.oneplusyou.com/q/img/bb_badges/countdown.jpg" alt="" style="display: none;" height="1" width="1" /></div>
 +
<div class="miniContainer">
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work">
 +
<img src="https://static.igem.org/mediawiki/2010/7/73/UofLNotebookbutton.jpg" width="80"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols">
 +
<img src="https://static.igem.org/mediawiki/2010/9/91/UofLprotocolsbutton.jpg" width="60"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Calendar">
 +
<img src="https://static.igem.org/mediawiki/2010/7/73/UofLcalendar.jpg" width="60"/>
 +
</a>
 +
</th>
 +
 
 +
<th>
 +
<div class="miniBar">
 +
<div class="countdown"><object type="application/x-shockwave-flash" data="http://www.oneplusyou.com/bb/files/countdown/countdown.swf?co=FFFFFF&bgcolor=000000&date_month=11&date_day=05&date_year=0&un=THE IGEM JAMBOREE&size=normal&mo=11&da=05&yr=2010" width="300" height="100"><param name="movie" value="http://www.oneplusyou.com/bb/files/countdown/countdown.swf?co=FFFFFF&bgcolor=000000&date_month=11&date_day=05&date_year=0&un=THE IGEM JAMBOREE&size=normal&mo=11&da=05&yr=2010" /><param name="bgcolor" value="#000000" /></object><img src="http://www.oneplusyou.com/q/img/bb_badges/countdown.jpg" alt="" style="display: none;" height="1" width="1" /></div>
 +
<div class="miniContainer">
 +
</th>
 +
<tr>
 +
</table>
 +
</center>
 +
</body>
 +
</html>
 +
 
 +
<hr>
 +
 
 +
<html>
 +
<center>
 +
<font color="white">Here you can check out the work we have done in the lab, click on a month to take a look!
 +
</center>
 +
</html>
 +
 
 +
<html>
 +
<body>
 +
<center>
 +
<table border="0"  width="50%"  style="background-color:#000000">
 +
 
 +
<tr>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/April">
 +
<img src="https://static.igem.org/mediawiki/2010/8/8a/UofLapril.jpg" width="80"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/May">
 +
<img src="https://static.igem.org/mediawiki/2010/7/7b/UofLmaybutton.jpg" width="60"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/June">
 +
<img src="https://static.igem.org/mediawiki/2010/8/80/UofLjunebutton.jpg" width="60"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/July">
 +
<img src="https://static.igem.org/mediawiki/2010/5/53/UofLjulybutton.jpg" width="60"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/August">
 +
<img src="https://static.igem.org/mediawiki/2010/1/15/UofLaugustbutton.jpg" width="60"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/September">
 +
<img src="https://static.igem.org/mediawiki/2010/4/4d/UofLseptemberbutton.jpg" width="60"/>
 +
</a>
 +
</th>
 +
 
 +
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/October">
 +
<img src="https://static.igem.org/mediawiki/2010/4/4e/UofLoctoberbutton.jpg" width="60"/>
 +
</a>
 +
</th>
 +
 
 +
 
 +
 
 +
<tr>
 +
</table>
 +
</center>
 +
</body>
 +
</html>
 +
<hr>
 +
 
 +
<BLOCKQUOTE>
 +
 
 +
=<font color="white">April=
 +
==<font color="white">April 13/2010==
 +
(In the Lab: JV, AS)<br>
 +
<b>Objective:</b> Test Restriction Endonucleases for Activity<br>
 +
<b>Relevant Information:</b><br>
 +
Endonucleases available
 +
<table><table border="3">
 +
<tr><td><b>Endonuclease</b></td><td><b>Optimal Buffer(**)</b></td><td><b>Other Buffers</b></td></tr>
 +
<tr><td>EcoRV</td><td>None</td><td>2xT(100%); O,G(50-100%)</td></tr>
 +
<tr><td>EcoRI</td><td>Red</td><td>O(100%);R(100%)(*);2xT(100%)</td></tr>
 +
<tr><td>BcuI/SpeI</td><td>Tango</td><td>B(50-100%);G(50-100%)</td></tr>
 +
<tr><td>XbaI</td><td>Tango</td><td>B,G,2xT(50-100%)</td></tr>
 +
<tr><td>PstI</td><td>Orange</td><td>R(100%); B,G,T,2xT(50-100%)</td></tr>
 +
<tr><td>DpnI</td><td>Tango</td><td>B,G(100%): O,R,2xT(50-100%)</td></tr>
 +
</table>
 +
(*)Star Activity<br>
 +
(**)Optimal Buffer from Fermentas<br><br>
 +
Use pUC19 plasmid as test, it has cut sites for EcoRI, PstI, XbaI (unsure about BcuI/SpeI, DpnI but will try anyways), and none for EcoRV <br>
 +
<u>Red Buffer:</u> EcoRI, PstI, Control (No Enzyme)<br>
 +
<u>Tango Buffer:</u> BcuI/SpeI, XbaI, DpnI, Control (No Enzyme)<br><br>
 +
<b>Methods:</b>
 +
Set up Master Mixes:
 +
<table><table border="3">
 +
<tr><td><b>Red MM</b></td><td>per tube (&micro;L)</td><td>Total (&micro;L)</td></tr>
 +
<tr><td>MilliQ H<sub>2</sub>0</td><td>13.75</td><td>55</td></tr>
 +
<tr><td>Red Buffer (10x)</td><td>2</td><td>7</td></tr>
 +
<tr><td>pUC19 (10pg/&micro;L)</td><td>2</td><td>7</td></tr>
 +
<tr><td><b>Total</b></td><td>19.75</td><td>69</td></tr>
 +
</table><br>
 +
<table><table border="3">
 +
<tr><td><b>Tango MM</b></td><td>per tube (&micro;L)</td><td>Total (&micro;L)</td></tr>
 +
<tr><td>MilliQ H<sub>2</sub>0</td><td>13.75</td><td>55</td></tr>
 +
<tr><td>Tango Buffer (10x)</td><td>2</td><td>7</td></tr>
 +
<tr><td>pUC19 (10pg/&micro;L)</td><td>2</td><td>7</td></tr>
 +
<tr><td><b>Total</b></td><td>19.75</td><td>69</td></tr>
 +
</table><br>
 +
To each tube, add <b>19.75&micro;L</b> of master mix and <b>0.25&micro;L</b> of enzyme<br>
 +
Incubated reaction mixes at 37<sup>o</sup>C (Start:7:00pm; End:7:45pm)<br>
 +
Add 3.3&micro;L of 6x loading dye to each reaction mixture and load 10&micro;L final volume onto a 1% agarose (in TAE) gel.<br>
 +
Add 1&micro;L of 6x loading dye to 1&micro;L of GeneRuler 1kb ladder (at 0.5&micro;g/&micro;L)<br>
 +
Gel loading order as follows:<br>
 +
<table><table border="3">
 +
<tr><td><b>Lane</b></td><td><b>Sample</b></td></tr>
 +
<tr><td>1</td><td>1kb Ladder</td></tr>
 +
<tr><td>2</td><td>Tango Control</td></tr>
 +
<tr><td>3</td><td>DpnI (Tango)</td></tr>
 +
<tr><td>4</td><td>BcuI/SpeI (Tango)</td></tr>
 +
<tr><td>5</td><td>XbaI (Tango)</td></tr>
 +
<tr><td>6</td><td>EcoRI (Red)</td></tr>
 +
<tr><td>7</td><td>PstI (Red)</td></tr>
 +
<tr><td>8</td><td>Red Control</td></tr>
 +
<tr><td>9</td><td>Empty</td></tr>
 +
<tr><td>10</td><td>Empty</td></tr>
 +
</table><br>
 +
Ran gel at 100V for 1 hour<br>
 +
 
 +
<b>Results:</b> pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain). <br>
 +
<b>Conclusion:</b> Will have to re-run experiment with DNA that is present at high enough concentrations to visualize by ethidium bromide staining<br><br><br>

Latest revision as of 00:44, 18 October 2010



Feel free to look around our notebook!

Here you can check out the work we have done in the lab, click on a month to take a look!

April

April 13/2010

(In the Lab: JV, AS)
Objective: Test Restriction Endonucleases for Activity
Relevant Information:
Endonucleases available

EndonucleaseOptimal Buffer(**)Other Buffers
EcoRVNone2xT(100%); O,G(50-100%)
EcoRIRedO(100%);R(100%)(*);2xT(100%)
BcuI/SpeITangoB(50-100%);G(50-100%)
XbaITangoB,G,2xT(50-100%)
PstIOrangeR(100%); B,G,T,2xT(50-100%)
DpnITangoB,G(100%): O,R,2xT(50-100%)

(*)Star Activity
(**)Optimal Buffer from Fermentas

Use pUC19 plasmid as test, it has cut sites for EcoRI, PstI, XbaI (unsure about BcuI/SpeI, DpnI but will try anyways), and none for EcoRV
Red Buffer: EcoRI, PstI, Control (No Enzyme)
Tango Buffer: BcuI/SpeI, XbaI, DpnI, Control (No Enzyme)

Methods: Set up Master Mixes:

Red MMper tube (µL)Total (µL)
MilliQ H2013.7555
Red Buffer (10x)27
pUC19 (10pg/µL)27
Total19.7569

Tango MMper tube (µL)Total (µL)
MilliQ H2013.7555
Tango Buffer (10x)27
pUC19 (10pg/µL)27
Total19.7569

To each tube, add 19.75µL of master mix and 0.25µL of enzyme
Incubated reaction mixes at 37oC (Start:7:00pm; End:7:45pm)
Add 3.3µL of 6x loading dye to each reaction mixture and load 10µL final volume onto a 1% agarose (in TAE) gel.
Add 1µL of 6x loading dye to 1µL of GeneRuler 1kb ladder (at 0.5µg/µL)
Gel loading order as follows:

LaneSample
11kb Ladder
2Tango Control
3DpnI (Tango)
4BcuI/SpeI (Tango)
5XbaI (Tango)
6EcoRI (Red)
7PstI (Red)
8Red Control
9Empty
10Empty

Ran gel at 100V for 1 hour

Results: pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain).

Conclusion: Will have to re-run experiment with DNA that is present at high enough concentrations to visualize by ethidium bromide staining


Retrieved from "http://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/April"